MEMS-based calorimeter, fabrication, and use thereof

ABSTRACT

MEMS-based calorimeter including two microchambers supported in a thin film substrate formed on a polymeric layer is provided. The thin film substrate includes a thermoelectric sensor configured to measure temperature differential between the two microchambers, and also includes a thermally stable and high strength polymeric diaphragm. Methods for fabricating the MEMS-based calorimeter, as well as methods of using the calorimeter to measure thermal properties of materials, such as biomolecules, or thermodynamic properties of chemical reactions or physical interactions, are also provided.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US2014/041181, filed Jun. 5, 2014, and claims priority from U.S.Provisional Application Nos. 61/831,472, filed Jun. 5, 2013; 61/915,995,filed Dec. 13, 2013; and 62/007,806, filed Jun. 4, 2014. The disclosureof each of which is hereby incorporated by reference herein in itsentireties.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under DBI-0650020 andCBET-0854030, both awarded by the National Science Foundation. Thegovernment has certain rights in this invention.

BACKGROUND

Differential scanning calorimetry (DSC) is a thermoanalytical techniquethat measures heat generated or required in thermally active processesas the temperature of a sample is varied. When applied to biochemicalsystems, DSC can provide a label-free method to determine thethermodynamic properties of a wide variety of biomolecular interactionsand conformational transition. DSC instruments, however, can becumbersome and require large sample consumption, which has hindered thewidespread application of DSC to biomolecular characterization.

Microelectromechanical Systems (MEMS) are small integrated devices orsystems that combine electrical and mechanical components in very smallmechanical devices. MEMS technology is based on fabrication technologiesthat can realize miniaturization, multiplicity, and microelectronics.

Some currently available MEMS calorimeters provide solid- or gas-phaseor droplet-based detections. However, it can be difficult to properlyhandle liquid samples in a well-defined environment in the currentlyavailable MEMS calorimeters.

Flow-through and continuous-flow MEMS calorimeters integratemicrofluidic chambers or channels as biological reactors. These devicescan provide controlled fluidic environments and can allow easyintegration with other microfluidic functionalities or thermal sensingconfigurations for biochemical thermodynamic investigations. However,these devices can still require a large amount of samples while beinglimited by significant convective heat leakage due to the continuousflow.

In addition, calibrating existing MEMS DSC devices can be complicateddue to a lack of integrated heating elements and temperature sensing.Temperature-modulated calorimetry (AC calorimetry) involves calorimetricmeasurements under small temporally periodic temperature variations.Such temperature modulation can allow thermal relaxation ofbiomolecules, and thus AC calorimetry can detect biomolecularinteractions under quasi-equilibrium conditions, and allow thebiochemical reaction signal to be extracted at the modulation frequencyin the face of broad-band background noise. However, these chips caninvolve thin solid films and operating parameters which are not suitablefor biomolecular characterization in solution phase.

Isothermal titration calorimetry (ITC) can measure heat generated orrequired for a biochemical reaction as a function of the molar reactantratio, and has been used in applications such as drug discovery andbiotherapeutic development. However, conventional ITC instruments canhave complicated structural designs, slow thermal response, and largesample and reagent consumption.

SUMMARY

In accordance with one aspect of the disclosed subject matter, amicrodevice is provided. The microdevice includes a first thermallyisolated microchamber, a second thermally isolated microchamber, and athin film substrate. The first and second microchambers can be a samplechamber and a reference chamber, respectively. The sample and referencechambers can be identical in volume and configuration, and arranged sideby side, each supported on the thin film substrate. The thin filmsubstrate can include a thermoelectric sensor located under each of thesample and reference chambers and configured to measure the temperaturedifferential between the sample and reference chambers. The thin filmsubstrate can also include a polymeric diaphragm made of a materialhaving a glass transition temperature and a thermal decompositiontemperature above the temperature range for the desired measurements.For example, in accordance with certain embodiments, the glasstransition temperature of the material can be greater than 150° C. andthe thermal decomposition temperature can be greater than 250° C. In anexemplary embodiment, the diaphragm material can be polyimide.

In some embodiments of the microdevice, the thermoelectric sensorincludes a thermocouple having a thermoelectric sensitivity of greaterthan 80 μV/° C. In other embodiments, the thermoelectric sensor isconfigured as a thin-layer thermopile including a plurality of elongatedsegments of dissimilar materials, adjacent segments of dissimilarmaterials being joined together at opposite ends, thereby formingthermocouple junctions. For example, the dissimilar thermoelectricmaterials can include n-type and p-type bismuth telluride, and n-typeand p-type antimony telluride. In one exemplary embodiment, thethermoelectric materials are antimony-bismuth (Sb—Bi).

The material of the polymeric diaphragm for the thin film substrate canhave a tensile strength and Young's modulus sufficient for structuralintegrity. For example, in certain embodiments the material can have atensile strength of greater than 55 MPa and a Young's Modulus greaterthan 500 MPa. For example, the polymeric diaphragm can be made from amaterial such as but not limited to polyimide, parylene, polyester, andpolytetrafluoroethylene. In one embodiment, the polymeric diaphragm ismade from polyimide.

In certain embodiments, the thin film substrate of the microdevice canfurther include a first microheater and a first temperature sensor, eachaligned under the first thermally isolated microchamber; and a secondmicroheater and a second temperature sensor, each aligned under thesecond thermally isolated microchamber. In such embodiments, thethermocouple junctions of the thermoelectric sensor can be located nearthe center of each of the first and second thermally isolatedmicrochambers, and vertically aligned with the first temperature sensorand the second temperature sensor, respectively. The microheaters andthe temperature sensors can be in a form of a thin layer of depositedmetal/alloy or metals/alloys impregnated in the thin film substrate. Themicroheaters can be patterned to provide uniform heating for themicrochambers.

In some embodiments, the chambers of the microdevice are defined by asurrounding wall made from a polymer such as SU-8, parylene,polycarbonate, polyether ether ketone (PEEK), or polydimethylsiloxane(PDMS). The thin film substrate of the microdevice can include a toplayer made from a mixture of the polymer of the surrounding wall and thematerial from which the polymeric diaphragm is made.

In some embodiments, the microdevice further includes a firstintroduction channel and a second introduction channel. Each of thefirst introduction channel and a second introduction channel can beconfigured to provide passive chaotic mixing for a solution flowingthrough the introduction channel. For example, the first and/or thesecond introduction channel can include a portion having a serpentineshape, and can further include internal ridges sufficient for creatingturbulence in the solution flowing through the first or the secondintroduction channel.

In accordance with another aspect of the disclosed subject matter, amethod of determining a thermal property of an analyte is provided. Asample material containing an analyte is provided in the sample chamber,and a reference material not containing the analyte is provided in thereference chamber. A thermal enclosure enclosing the microdevice isheated at a predetermined temperature scanning rate. A thermal propertyof the analyte can be determined based on the measured temperaturedifferential between the sample chamber and reference chamber.

In some embodiments of the method, a temporally periodic variation inthe heating power can be provided during the heating of the thermallyisolating enclosure. Providing the temporally periodic variation in theheating power can be performed using the microheaters of the microdevicefor temporally modulated heating. The temporal modulation of the heatingcan be controlled by a waveform generator.

In accordance with another aspect of the disclosed subject matter, amethod of determining heat involved in a reaction between at least twosubstances is disclosed. A sample solution containing a mixture of afirst substance and a second substance is provided in the samplechamber, a reference solution is provided in the reference chamber. Athermal enclosure enclosing the microdevice is maintained at a constanttemperature. The heat involved in the reaction between the firstsubstance and the second substance at the given temperature can bedetermined based on the measured temperature differential between thesample chamber and reference chamber.

The reaction between the first and second substances can be a chemicalreaction or a physical binding system, for example, ligand-proteinbinding. The thermal enclosure temperature can be varied such that theheat involved in the reaction can be determined at differenttemperature. Likewise, the concentration ratio between the twosubstances can also be varied such that reaction stoichiometry can bedetermined by the heat measured at different concentration ratios. Thesample and/or reference solution can be fed into the respective chambersthrough an introduction channel as described above, which can providepassive mixing.

In an exemplary embodiment, a microelectromechanical systems-basedcalorimetric device for characterization of biomolecular interactionsincludes a first micromixer, a second micromixer, a thermally-isolatedreaction chamber, a thermally-isolated reference chamber, and athermoelectric sensor. The thermally-isolated reaction chamber is influid contact with the first micromixer. The thermally-isolatedreference chamber is in fluid contact with the second micromixer. Thethermoelectric sensor is configured to measure at least one temperaturemetric associated with reaction chamber and the reference chamber.

The first and second micromixers can be passive chaotic micromixers. Forexample, the first and second micromixers can be formed from aserpentine channel with herringbone shaped ridges on the ceilingthereof. The device can further include a first inlet a second inlet influid contact with the first micromixer, and a third inlet and a fourthinlet in fluid contact with the second micromixer.

The reaction chamber and reference chamber can be polymericmicrochambers such as polydimethylsiloxane microchambers. The reactionchamber and reference chamber can be serpentine chambers. The reactionchamber and reference chamber can be disposed on a diaphragm such as apolyimide diaphragm that serves as a base for the reaction chamber andthe reference chamber.

In accordance with an exemplary embodiment of the disclosed subjectmatter, the thermoelectric sensor can be a thermopile. The thermopilecan be, for example, an antimony-bismuth thermopile. A first thermopilejunction can be located on a first side of the reaction chamber, while asecond thermopile can be located on the first side of the referencechamber.

The reaction chamber and reference chamber can be surrounded by an aircavity. In accordance with an exemplary embodiment of the disclosedsubject matter, the air cavity can include a serpentine channel. Thedevice can further include a temperature sensor and a heater for thereaction chamber and the reference chamber.

In accordance with an exemplary embodiment of the disclosed subjectmatter, the at least one temperature metric can be a differentialtemperature between the reaction chamber and the reference chamber. Inother embodiments, the at least one temperature metric can be atemperature of the reaction chamber and a temperature of the referencechamber.

The disclosed subject matter further provides microelectromechanicalsystems-based methods for characterization of a biomolecular interactionbetween a first solution and a second solution. In one example, a methodincludes mixing the first solution and the second solution to form areaction solution, mixing the first solution and a buffer solution toform a reference solution, and measuring a differential temperaturebetween a reaction chamber containing the reaction solution and areference chamber containing the reference solution. The differentialtemperature can be measured using a thermoelectric sensor such as athermopile on the microelectromechanical systems-based device.

In accordance with an exemplary embodiment of the disclosed subjectmatter, micromixers on the microelectromechanical systems-based device(e.g., passive chaotic micromixers) can be used to mix the first andsecond solutions.

The method can further include computing a differential power based atleast in part on the differential temperature. At least onethermodynamic reaction parameter can be calculated based at least inpart on the differential power. The thermodynamic reaction parameter canbe, for example, an equilibrium binding constant, a stoichiometry, or amolar enthalpy change.

A baseline temperature differential between the reaction chamber and thereference chamber can be measured prior to the introduction of thereaction solution and the reference solution. The baseline temperaturedifferential can then be subtracted from the differential temperaturefor error correction. The device can also be calibrated using an on-chipheater.

The disclosed subject matter further provides microelectromechanicalsystems-based calorimetric devices for characterization of biomolecularreactions. In an exemplary embodiment, a device includes first mixingmeans for mixing a first solution and a second solution, second mixingmeans for mixing the first solution and a buffer solution, athermally-isolated reaction chamber in fluid contact with the firstmixing means, a thermally-isolated reference chamber in fluid contactwith the second mixing means, and detection means for measuring adifferential temperature between the reaction chamber and the referencechamber. The device can further include computing means for computing adifferential power based at least in part on the differentialtemperature, and calculating means for calculating at least onethermodynamic reaction parameter based at least in part on thedifferential power.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1C depict a schematic of a microdevice according to someembodiments of the disclosed subject matter, in top (FIG. 1A), isometric(FIG. 1B), and sectional (FIG. 1C) views.

FIG. 2A-FIG. 2E depict a procedure for the fabrication of themicrodevice according to some embodiments of the disclosed subjectmatter.

FIG. 3 is a schematic diagram illustrating the principle of ACdifferential scanning calorimetry according to the disclosed subjectmatter.

FIG. 4A and FIG. 4B are schematics of a microdevice according to someembodiments of the disclosed subject matter for isothermal titrationcalorimetry.

FIG. 5A-FIG. 5D Are images of a microdevice fabricated according to oneembodiment of the disclosed subject matter: (FIG. 5A) the PDMS housingstructure and air gap; (FIG. 5B) the solid substrate; (FIG. 5C) thethermopile, integrated microheater and temperature sensor embedded inthe thin film substrate; and (FIG. 5D) the thermopile junctions.

FIG. 6A is a schematic diagram of a testing setup for a calorimetricmeasurement using a microdevice according to some embodiments of thedisclosed subject matter.

FIG. 6B shows the details of a custom-built, temperature-controlledthermal enclosure as compared with a schematic diagram of the thermalenclosure, according to some embodiments the disclosed subject matter.

FIG. 7 is a plot showing the thermopile output voltage from amicrodevice according to some embodiments the disclosed subject matterin response to constant temperature difference between the thermopile'shot and cold junctions.

FIG. 8 is a plot showing a steady-state response (in terms of thermopileoutput voltage) from a microdevice according to some embodiments thedisclosed subject matter in response to constant differential powerbetween the two chambers of the microdevice.

FIG. 9 is a plot showing the transient response of a microdeviceaccording to some embodiments the disclosed subject matter with respectto a step differential power.

FIG. 10 is a plot showing the output of a microdevice according to someembodiments the disclosed subject matter as a function of temperature ina temperature scan during which the unfolding of lysozyme occurs.

FIG. 11A and FIG. 11B are plots showing partial specific heat capacity(FIG. 11A) and change of molar enthalpy (FIG. 11B) as a function oftemperature during the unfolding of lysozyme, as measured by themicrodevice according to some embodiments the disclosed subject matter.

FIG. 12A and FIG. 12B are plots showing the output of a microdeviceaccording to some embodiments the disclosed subject matter (10 a) andchange of molar enthalpy as a function of temperature during theunfolding of lysozyme at varying temperature scanning rates.

FIG. 13 is a schematic diagram showing an experiment setup for AC-DSCmeasurements according to some embodiments of the disclosed subjectmatter.

FIG. 14 is a plot showing the output voltage of the thermopile of amicrodevice according to some embodiments of the disclosed subjectmatter in response to constant differential temperature between its hotand cold junctions.

FIG. 15 is a plot showing a steady-state response (in terms ofthermopile output voltage) from a microdevice according to someembodiments the disclosed subject matter in response to constantdifferential power between the two chambers of the microdevice.

FIG. 16 is a plot showing the transient response of a microdeviceaccording to some embodiments the disclosed subject matter with respectto a step differential power.

FIG. 17 is a plot showing the frequency dependence of thermopile voltage(baseline subtracted) of a microdevice according to some embodiments thedisclosed subject matter, where the sample chamber was filled withlysozyme (20 mg/mL) and the reference chamber was filled with 0.1 MGlycine-HCl buffer (pH 2.5).

FIG. 18A and FIG. 18B are plots showing changes in (FIG. 18A) amplitude,and (FIG. 18B) phase, of the thermopile voltage as a function oftemperature during the unfolding of lysozyme at different lysozymeconcentrations and AC modulating frequencies, as measured on amicrodevice according to some embodiments the disclosed subject matter.

FIG. 19 is a plot showing specific heat capacity of lysozyme as afunction of temperature during the unfolding of lysozyme at differentlysozyme concentrations and AC modulation frequencies, as measured on amicrodevice according to some embodiments the disclosed subject matter.

FIG. 20 is a plot showing a comparison between a DC-DSC measurement andan AC-DSC measurement for specific heat capacity of lysozyme during itsunfolding process, using a microdevice according to some embodiments thedisclosed subject matter.

FIG. 21A and FIG. 21B are images of certain elements of a microdevicefor isothermal titration calorimetry according to some embodiments ofthe disclosed subject matter.

FIG. 22 is a schematic diagram showing the experimental setup forisothermal titration calorimetry according to some embodiments of thedisclosed subject matter.

FIG. 23A and FIG. 23B are plots showing calibration results of amicrodevice according to some embodiments of the disclosed subjectmatter for performing isothermal titration calorimetry: transientresponse to a step differential power (FIG. 23A), and steady-stateresponse to a constant differential power (FIG. 23B).

FIG. 24 is a plot showing a time-resolved output of a microdeviceaccording to some embodiments of the disclosed subject matter uponintroduction of 5 mM 18-C-6 and 4 mM BaCl₂ (each 0.5 μL), compared withmeasurement of 5 mM 18-C-6 titrated by DI water (plotted with a 4 μVoffset for clarity).

FIG. 25A and FIG. 25B are plots for the output of a microdeviceaccording to some embodiments of the disclosed subject matter in theisothermal titration calorimetric measurement of the binding of 5 mM18-C-6 and BaCl₂ at continuous injections with a series of molar ratios(FIG. 25A); and calculated heat of the binding of 18-C-6 and BaCl₂ as afunction of molar ratio. The fitted curve is based on a one-site bindingmodel.

FIG. 26 is a plot showing the biochemical heat of binding of 18-C-6 andBaCl₂ as a function of molar ratio at temperatures of 23 and 35° C., ascalculated from the output of a microdevice according to someembodiments of the disclosed subject matter.

FIG. 27 depicts a top view of a schematic of a microdevice in accordancewith an exemplary embodiment of the disclosed subject matter.

FIG. 28 is a diagram illustrating an electronic circuit that can becoupled to a microdevice in accordance with an exemplary embodiment ofthe disclosed subject matter.

FIG. 29 is a flowchart illustrating a method for measuring adifferential temperature and characterizing a reaction in accordancewith an exemplary embodiment of the disclosed subject matter.

FIG. 30 illustrates a comparison of measurements of the time-resolvedthermopile voltage upon introduction of 4 mM BaCl₂ and 5 mM 18-C-6 (each0.5 μL) in the reaction chamber, and the signal upon introduction ofsterile water and 5 mM 18-C-6 (also each 0.5 μL) in accordance with anembodiment of the disclosed subject matter.

FIG. 31 illustrates the baseline-subtracted device output of a devicefor characterizing biochemical reactions in accordance with oneembodiment of the disclosed subject matter.

FIG. 32 illustrates the calculated reaction heat derived from the outputvoltage measurements in accordance with one embodiment of the disclosedsubject matter.

FIG. 33 shows a comparison between reaction parameters obtained inaccordance with an embodiment of the disclosed subject matter andpublished data reflecting reaction parameters obtained using commercialcalorimeters.

FIG. 34 illustrates device output exhibited titration-dependent spikesin correspondence to the molar ratio at varying molar ratios inaccordance with an embodiment of the disclosed subject matter.

FIG. 35 shows a second comparison between reaction parameters obtainedin accordance with an embodiment of the disclosed subject matter andpublished data reflecting reaction parameters obtained using commercialcalorimeters.

FIG. 36 depicts an isometric view of a schematic of a microdevice inaccordance with an exemplary embodiment of the disclosed subject matter.

FIG. 37A-FIG. 37B depicts top and isometric views of a schematic of amicrodevice in accordance with an exemplary embodiment of the disclosedsubject matter.

FIG. 38A-FIG. 38E depicts a process for fabricating a microdevice inaccordance with an exemplary embodiment of the disclosed subject matter.

FIG. 39 is a graph showing the change in heat capacity versustemperature as measured in accordance with one embodiment of thedisclosed subject matter as compared with calculated values.

FIG. 40A-FIG. 40D shows temperature distribution within a microfluidicstructure in accordance with one embodiment of the disclosed subjectmatter.

FIG. 41 shows transient responses to a unit step power in accordancewith one embodiment of the disclosed subject matter.

FIG. 42 is a graph showing thermopile differential voltage as a functionof temperature, corrected by baseline subtraction, as measured atvarying concentrations from 1 to 20 mg/mL in accordance with oneembodiment of the disclosed subject matter.

FIG. 43 is a graph showing total enthalpy change per mole of lysozomeunfolding as a function of temperature at different lysozomeconcentrations in accordance with one embodiment of the disclosedsubject matter.

DETAILED DESCRIPTION

In accordance with one aspect of the disclosed subject matter, amicrodevice is provided. The microdevice includes a first thermallyisolated microchamber, a second thermally isolated microchamber, and athin film substrate. The first and second microchambers are alsoreferred herein as the sample chamber and reference chamber,respectively. The sample and reference chambers can be identical involume and configuration, and arranged side by side. In accordance withan exemplary embodiment of the disclosed subject matter, the sample andreference chambers can have a circular configuration. However, a widevariety of geometric configurations can be used in accordance with thedisclosed subject matter. The sample and reference chambers can each besupported on the thin film substrate. The thin film substrate caninclude a thermoelectric sensor located under each of the sample andreference chambers and configured to measure the temperaturedifferential between the sample and reference chambers.

FIGS. 1a-1c depict an illustrative embodiment of the microdevice of thedisclosed subject matter. The microdevice is also referred to as MEMSDSC device herein. The microdevice 100 includes two identicalmicrochambers 110 and 120, which can hold sample and reference materialsfor calorimetric measurements. For easy reference, these microchambersare also referred to herein as the sample chamber and reference chamber,respectively, and collectively, “calorimetric chambers,” or simply“chambers” Each of the sample and reference chambers is connected to aninlet port (111, 121) and an outlet port (112, 122) by microfluidicchannels. The material for the housing of the chambers (140) can be madeof any material suitable for microfabrication and thermal isolation. Incertain embodiments, Polydimethylsiloxane (PDMS) is selected as thematerial to fabricate the calorimetric chambers for its ease offabrication and packaging as well as biocompatibility. However, othermaterials suitable for microfabrication and thermal isolation can beused without departing from the scope of the disclosed subject matter.For example, a material having sufficient thermal stability within thetemperature range of interest (e.g., −10° C. to 90° C.), reasonablystrong bonding with the substrate surface, and minimized adsorption ofmacromolecules (e.g., proteins) can be used, The microchambers can beformed from polymers such as SU-8, parylene, polycarbonate, andpolyether ether ketone (PEEK).

Each of the microchambers can be thermally isolated. For example, theair cavities (130) in FIG. 1a provide thermal isolation for thechambers. Air cavities can be formed from the same material used forfabrication of the microchambers. In accordance with an exemplaryembodiment of the disclosed subject matter, the air cavities (130) canbe formed from polydimethylsiloxane. However, other thermal isolationtechniques can also be used as known in the art. For example, themicrochambers can be thermally isolated by residing on a freestandingstructure constructed from materials such as a polymeric material havinglow thermal conductivity. In order to further isolate the microchambersfrom the ambient environment, the microdevice 100 can be enclosed by athermal enclosure (e.g., the microdevice 100 can be placed in a vacuumto minimize thermal energy dissipation to the ambient environment).

As shown in FIG. 1b , the microchambers (110, 120) are supported on athin film substrate (150). The thin film substrate (150) along with aircavities (130) surrounding the chambers, provide thermal isolation thatenables sensitive calorimetric measurements. The thin film (150) caninclude multiple polymeric layers or diaphragms (151, 152, 153). Thelayers 151, 152, 153 are integrated as shown in FIG. 1C, but for purposeof illustration, are shown in FIG. 1B as separate layers. Both of layers151 and can be made from a material have good thermal isolationproperty, as well as thermal and mechanical stability to withstand thethermal cycles required by repeated calorimetric measurements. Inparticular embodiments, the polymeric diaphragm can be made of amaterial having a glass transition temperature greater than 150° C. andthermal decomposition temperature greater than 250° C. For example, thematerial can be polyimide, parylene, polyester, SU-8, PDMS, andpolytetrafluoroethylene, etc. The polymeric diaphragm can have a tensilestrength of greater than 55 MPa, and/or Young's Modulus greater than 500MPa. In particular embodiments, polyimide is selected as the diaphragmmaterial because of to its excellent mechanical stiffness (Young'smodulus: 2.5 GPa) and thermal stability (glass transition temperature:285° C.).

To improve the adhesion between the housing material and the thin filmsubstrate, an interfacing layer 153 can be made from a mixture of thematerial for layer 151 and/or 152, e.g., a mixture of polyimide/PDMS.The thin film substrate can be supported on another solid substrate(160), e.g., a silicon wafer. To improve thermal isolation, the solidsubstrate in the area underneath the bottom side of the thin filmsubstrate corresponding to a cross section of each of the chambers canbe removed, such that the portion of the thin film substrate under eachof the chambers does not contact the solid substrate (i.e., it onlycontacts air, which is believed the best thermal insulator).

The microdevice can further include a thermoelectric sensor. Athermoelectric sensor can be coated on, embedded, or otherwise includedin the thin film substrate and configured to measure the temperaturedifferential between the two chambers. For example, a thin layer ofthermopile (170) can be included between the polymeric layers (152,153). As illustrated in FIGS. 1B and 1C, the thermopile can include aplurality of elongated segments of dissimilar materials, where adjacentsegments of dissimilar materials are joined together at opposite ends,thereby forming thermocouple junctions (171 and 172). The thermocouplejunctions underneath each chamber can be aligned to the central axis ofeach chamber. The material for the thermopile can include a variety ofdissimilar pairs of metals, e.g., antimony-bismuth (Sb—Bi), or otherpairs of materials providing high thermoelectric efficiency, such asn-type and p-type bismuth telluride, and n-type and p-type antimonytelluride. For example, the thermoelectric sensor can have athermoelectric sensitivity of greater than 80 μV/° C. per thermocouple.In particular embodiments, antimony (Seebeck coefficient: 43 μV/K) andbismuth (Seebeck coefficient: −79 μV/K) are selected for the thermopilematerial due to their high thermoelectric sensitivities and ease offabrication. A wide variety of metals, semiconductors, and theircompounds, including chrome, nickel, bismuth, antimony, bismuthtelluride, and antimony telluride, can be used in fabricating thethermopile.

In accordance with another embodiment of the disclosed subject matter,the thermoelectric sensor can include a sample chamber thermoelectricsensor and a reference chamber thermoelectric sensor, each of whichmeasures the absolute temperature of the reaction in the respectivemicrochambers. The differential temperature can then be determined bycalculating the different between the temperatures measured by thethermoelectric sensors. The thin film substrate can further include twosets of microheaters (180) and temperature sensors (190) which arealigned underneath the two chambers (110, 120), respectively. Forexample, the microdevice 100 can include an integrated tin-filmresistive micro-temperature sensor and heater. The temperature sensors(190) can monitor the chamber temperatures in real time, and themicroheaters (180) can provide heating to the chambers to generate aconstant differential power for calorimetric calibration. For purposesof calibration, Joule heating can be generated by passing an electricalcurrent through the microheater. The local temperature can then bedetermined by the temperature sensor based on a calibrated relationshipbetween the temperature and the electrical resistance.

Both of the microheaters (180) and temperature sensors (190) can beembedded in the thin film, but vertically away and insulated from thethermopile (170). For example, the microheaters (180) and temperaturesensors (190) can be embedded between layers 151 and 152. The contactpad (195) for the temperature sensor and the contact pad for themicroheaters (185) can extend outside of the chamber housing structurefor external electrical connection. Although shown in FIGS. 1b and 1c assituated on the same layer, the microheaters (180) and the temperaturesensors (190) can also be situated on different layers. For precisetemperature sensing, particularly in device calibration, the thermopilejunctions (171, 172) can be aligned with the temperature sensors (190).The microheaters (180) can be patterned in a way to provide uniformheating of the chambers, for example, in a meandering pattern underneaththe bottom area of the chambers. The material of the microheaters can bechosen from a variety of metals or metal alloys, for example,chromium/gold (Cr/Au).

In one embodiment, the microdevice illustrated in FIGS. 1A-1C can befabricated by a procedure as outlined below. A solid substrate (160),such as silicon wafer, is provided. In accordance with otherembodiments, a flexible layer can be used as a substrate rather than asilicon layer. The flexible layer can be, for example, a polymeric layersuch as a layer of Kapton film A polymeric diaphragm (151), e.g., apolyimide film, can be coated on the solid substrate, e.g., byspin-coating (FIG. 2A). A pair of cavities (165) can be etched using,for example, tetramethylammonium hydroxide (TMAH) into the backside ofthe solid substrate in the areas that correspond to the calorimetricchambers. After the curing of the polymeric diaphragm, microheaters(180) and temperature sensors (190) can be deposited by thermalevaporation of a metal or metal alloy, e.g., Cr/Au. This is followed bycoating another polymeric diaphragm (152) on top of the microheaters andtemperature sensors (FIG. 2B). Subsequently, the thermoelectric sensor(170), e.g., a thermopile, can be thermally evaporated and patternedusing a standard lift-off process, and the thermoelectric sensor isfurther coated by another polymeric layer (153), e.g., a layercontaining polyimide-PDMS mixture (FIG. 2C). The chamber housingstructure (140) can then be fabricated, e.g., from PDMS usingmicromolding techniques on top of the thin film substrate, therebyforming the calorimetric chambers (FIG. 2D). Microfluidic structure suchas microchannels connecting the chambers to the inlet and outlet ports(121/122) can also be fabricated (FIG. 2E). The residual silicon layeron the backside of the thin film can then be removed (FIG. 2E), therebyforming the freestanding thin film substrate portions under each of thechambers.

With reference to FIG. 27, a second embodiment of the microdevice inaccordance with the disclosed subject matter is shown. The device 2700includes a serpentine reference chamber (2702) and a serpentine samplechamber (2704). The reference chamber (2702) and sample chamber (2704)are thermally isolated by air cavities (2706). The microdevice 2700 alsoinclude a thermopile (2708). The use of serpentine microchambers (2702,2704) can allow for a greater number of thermopile junctions can improvethermal isolation.

The microdevice 2700 also includes one or more contact pads (2710,2712). The contact pads can provide an interface between the device andvarious electronic circuits. For example, contact pad 2710 can becoupled to the thermopile (2708). The adhesion between the ends of thethermopile (2708) and contact pad 2710 can be enhanced by surfaceroughening or chemical modification. A designed external packaging via aflip chip bonding method can also be implemented. The output of thethermopile (2708) is a voltage indicative of a differential temperaturebetween the reference chamber (2702) and the sample chamber (2704). Thecontact pad 2710 can also be coupled to an electronic circuit formeasuring and analyzing the output voltage. The term “coupled,” as usedherein, includes direct coupling such as direct electrical contact(e.g., through a soldered wire or alligator clip) as well as indirectcoupling, as through wireless communication.

An exemplary embodiment of an electronic circuit that can be coupled tocontact pad 2710 in accordance with the disclosed subject matter isillustrated in FIG. 28. The contact pad (2802) acts as the interfacebetween the microdevice and the one or more electronic circuits 2800.The contact pad can be coupled to a voltmeter (2804). The term“voltmeter,” as used herein, is intended to encompass any instrumentthat can be used to measure voltage, either directly or indirectly,including voltmeters and multimeters. The voltmeter (2804) can includeat least one processor.

The voltmeter (2804) can be coupled to a calculation device (2806). Thecalculation device (2806) includes one or more processors formed by oneor more electronic circuits. The calculation device (2806) can becoupled to a storage device (2808).

The calculation device (2806), as well as each of the componentsthereof, can be implemented in a variety of ways as known in the art.For example, each of the components of the calculation device can beimplemented using a single integrated processor. In another embodiment,each component can be implemented on a separate processor. One or morecomponents of the calculation device (2806) can be combined with thevoltmeter (2804) rather than being a separate device.

The at least one processor can include one or more electronic circuits.The one or more electronic circuits can be designed so as to implementthe disclosed subject matter using hardware only. Alternatively, theprocessor can be designed to carry out instructions specified bycomputer code stored in the storage device (2808). The storage devicecan be a hard drive, a removable storage medium, or any othernon-transitory storage media. Such non-transitory storage media canstore instructions that, upon execution, cause the at least oneprocessor to perform the methods disclosed herein.

The calculation device (2806) can include a number of components,including an adjustment component (2810) for adjusting the outputvoltage based on a baseline in output voltage, a thermal powerdifferential component (2812) for determining a thermal powerdifferential based on the output voltage, and a reactioncharacterization component (2814) for calculating thermodynamic reactionparameters based on the thermal power differential.

With further reference to FIG. 27, contact pad 2712 can be coupled to amicroheater and/or a temperature sensor. Contact pad 2712 can further becoupled to one or more electronic circuit for implementing the in-situtemperature monitoring and on-chip device calibration methods asdisclosed herein.

With reference to FIG. 36, a third embodiment of a microdevice inaccordance with the disclosed subject matter is shown. The device 3600includes a structure 3640 that forms microchambers. The microchamberscan be, for example, circular or serpentine in shape. Structure 3640 canbe a structure made of PDMS or another polymer, and can be supported bya thin film substrate 3650. Thin film substrate 3650 can includepolymeric layers 3651, 3652, and 3653. Polymeric layer 3652 can includea thermopile 3670. The thermopile 3670 can consist of Sb—Bi or otherthermoelectric junctions. Likewise, polymeric layer 3651 can includemicro-heaters 3680 and temperature sensors 3690.

The thin film substrate 3650 can be formed on a substrate 3660. Inaccordance with embodiment of the disclosed subject matter, thesubstrate can be a flexible material. For example, the flexible materialcan have a flexural modulus of at least about 1.0, at least about 1.2,at least about 1.4, at least about 1.4, at least about 1.6, at leastabout 1.8, or at least about 2.0.

In accordance with another embodiment, the substrate 3660 can be formedby a polymeric material. Polymeric materials suitable for use as asubstrate include, but are not limited to, polyimide, parylene,polyester, and polytetrafluoroethylene. The material can have a tensilestrength and Young's modulus sufficient for the structural integrity, aswell as having a glass transition temperature above the temperaturerange for the desired measurements. The thickness of the polymericsubstrate can be between about 5 μm and about 1000 or between about 10μm and about 500 μm. For example, in accordance with embodiments of thedisclosed subject matter, the thickness of the flexible substrate can beabout 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about50 μm, about 75 μm, about 100 μm, about 150 μm, about 200 μm, about 250μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, or about 500μm.

Microdevices formed on flexible layers such as polymeric layers canimprove robustness and fabrication yield due to low intrinsic stress ofthe polymer as compared to more fragile substrates such as a siliconsubstrate. In addition, the low thermal conductivity of the polymersubstrate can lead to enhanced thermal isolation of measurement samplesfor improved sensitivity. In addition, because the polymer substrate canbe flexible, deformation of the substrate can be permitted, which canallow the device to conform to non-planar surfaces. As such, the devicecan be used in applications that involve geometry with curvature.Devices with polymeric substrates can also be low cost and disposable,which can eliminate cross-contamination between samples.

With reference to FIG. 37, a fourth embodiment of a microdevice inaccordance with the disclosed subject matter is shown. The device 3700includes a structure 3740 that forms microchambers. The microchamberscan be, for example, circular or serpentine in shape. Structure 3740 canbe a structure made of PDMS or another polymer, and can be supported bya thin film substrate 3750. Thin film substrate 3750 can include layer3751 and polymeric layers 3752, and 3753. Polymeric layer 3752 caninclude a micro-heaters (not shown) and temperature sensors 3790.Likewise, layer 3751 can include thermopile 3770. The thermopile 3770can consist of Sb—Bi or other thermoelectric junctions. Layer 3751 canbe a flexible layer and/or a polymeric layer. The polymeric layer can beconstructed using the same materials and dimensions as the polymericlayer 3660 described above.

With reference to FIG. 38, in one embodiment, the microdeviceillustrated in FIG. 37 can be fabricated by a procedure as outlinedbelow. In FIG. 38(a), a layer 3802 can be reversibly bound to siliconwafer 3804. While the FIG. 38 will be described with reference to aflexible layer, it will be understood that a polymeric layer can also beused in accordance with the disclosed subject matter. The flexible layer3802 can be, for example, a polymeric layer such as a polyimide film(e.g., a Kapton film). The flexible layer can have a thickness of about12.5 μm. The flexible layer 3802 can be reversibly bound to the siliconwafer 3804 by an adhesive layer 3806 such as a spin-coated PDMS layer.The adhesive layer can have a thickness of about 20 μm. The adhesivelayer can then be cured.

In FIG. 38(b), a thermopile can be formed on the flexible layer 3802.For example, Sb and Bi can be thermally evaporated and patterned on thesubstrate using a standard lift-off process to form a 400-junctionthermopile 3808. In accordance with one embodiment, the antimony canhave a thickness of about 0.8 μm and the bismuth can have a thickness ofabout 1 μm. The thermopile can then be passivated with a spin-coatedpolyimide thin layer 3810. The thin layer 3810 can have a thickness ofabout 1.5 μm.

In FIG. 38(c), temperature sensors 3812 can be formed on the thin layer3810. For example, a chromium/gold tin film can be deposited andpatterned to form an on-chip temperature sensor 3812. The chromium/goldthin film can have a thickness of about 100 μm. Contact pads 3814 can beformed in a similar manner.

In FIG. 38(d), the device can be separated from the silicon wafer 3804.In accordance with some embodiments, the device can first be passivatedwith another thin layer 3816 such as a thin PDMS layer. The device canthen be mechanically released from the silicon wafer 3804. The adhesivelayer 3806 can also be removed during this process. In accordance withsome embodiments of the disclosed subject matter, the silicon wafer canbe reused for manufacture of subsequent microdevices.

In FIG. 38(e), microchambers can be formed. The formation ofmicrochambers can be performed in parallel with the removal of thesilicon wafer 3804. In accordance with one embodiment of the disclosedsubject matter, serpentine microfluidic channels can be formed in apolymeric layer 3818 (such as a PDMS layer) via soft lithography. Thepolymeric layer 3818 can be bonded to the microfluidic structure using,for example, oxygen plasma.

In accordance with another aspect of the disclosed subject matter, amethod of determining a thermal property of an analyte is provided. Themethod includes providing a microdevice as described above, providing athermal enclosure enclosing the microdevice; loading a sample materialcontaining an analyte into the first microchamber; loading a referencematerial into the second microchamber, the reference material does notcontain the analyte; heating the thermal enclosure at a predeterminedtemperature scanning rate; and determining a thermal property of theanalyte based on the measured temperature differential between the firstmicrochamber and the second microchamber. The microdevice and the methodof using the microdevice for calorimetric measurement are furtherdescribed in conjunction with each other in the Examples below. It isappreciated that the microdevice including any of the specific featuresdescribed below can be used in the method of using the microdevice, andvise versa.

In some embodiments of the above method, a temporally periodicvariation, or AC modulated heating, can be introduced to the referenceand sample materials during the heating of the thermal enclosure, asillustrated in FIG. 3. This can lead to temperature modulation, whichallows thermal relaxation of biomolecules, as well as allow thebiochemical reaction signal to be readily extracted at the modulationfrequency in the broad-band background noise. The temperature modulationcan be achieved by using the microheaters included in the thin filmsubstrate of the microdevice, controlled by a wave generator which canprovide different frequency, magnitude, and other parameters for theon-chip heating.

In accordance with another aspect of the disclosed subject matter, amethod of determining heat involved in a reaction between at least twosubstances is provided. The method includes: providing a MEMS device asdescribed above; providing a thermal enclosure enclosing themicrodevice; feeding a sample solution into the first thermally isolatedmicrochamber, wherein the sample solution is prepared by mixing a firstsubstance with a second substance; feeding a reference solution into thesecond thermally isolated microchamber, the reference solution does notcontain at least one of the first and the second substances; anddetermining the heat involved in the reaction between the firstsubstance and the second substance based on the measured temperaturedifferential between the sample chamber and the reference chamber.During the measurement, the temperature of the thermal enclosure (thatencloses the microdevice) can be maintained at a constant value. Thus,the method is also referred to as isothermal titration calorimetry(ITC). The reaction between the first and second substances can be achemical reaction or physical binding. Thus, the two substances can beany of the variety of chemicals, biomolecules or other molecules thatare reactive to each other, receptor-ligand, protein-enzyme, acid-base,etc., wherein the reaction between the two substances either generate,or absorb measurable heat.

An exemplary method for measuring a differential temperature andcharacterizing a reaction in accordance with an embodiment of thedisclosed subject matter is shown in FIG. 29. The method can includecalibrating the device, measuring a baseline in device output, mixingthe sample and the reactant, mixing the sample and the buffer, measuringa differential temperature, determining the thermal power, andcalculating the thermodynamic reaction parameters.

To begin, the calorimetric device can be calibrated at 2902. Forexample, calibration techniques known in the art are described in A MEMSDifferential-Scanning-calorimetric Sensor for ThermodynamicCharacterization of Biomolecules by Bin Wang and Qiao Lin, J.Microelectromechanical Systems 21:5, 1165-1171 (October 2012), which isincorporated by reference herein in its entirety for all purposes.

The baseline in device output can then be measured at 2904. For example,if a thermopile is used to measure the differential temperature, thethermopile output voltage in the absence of a reaction can be measured.This can be accomplished by introducing a mixture of sample and buffersolutions into each of the chambers. The baseline in device output canthen be stored in storage device 2808 as shown in FIG. 28 for futureuse.

A sample and a reactant can then be mixed at 2906. The sample and abuffer can be mixed substantially simultaneously at 2908. Mixing can beaccomplished using a passive chaotic mixer such as the one illustratedin FIG. 4a . Using the device 400, the sample can introduced into inlet431 and the reactant can be introduced into inlet 432. The sample and abuffer can be introduced in corresponding inlets to introduction channel440. The sample and the reactant are passively mixed in introductionchannel and deposited into the sample chamber 410. The sample and thebuffer are passively mixed in introduction channel 440 and depositedinto the reference chamber 420. Titration techniques known in the artfor use with Isothermal Titration calorimetry (ITC) can be used.

In accordance with an embodiment of the disclosed subject matter,titration on the MEMS device can be performed with a series of discretereactions, with each reaction having a specific molar ratio of thereactants. Liquid cartridge segments can be used for introduction ofreactants. For example, binding reagents in different concentrations canbe prepared while the sample is prepared in a fixed concentration. Assuch, the molar ratio can be varied with the volume of sample andbinding reagent maintained identical (e.g., 0.5 μL). The sample andbinding reagent can each be loaded in a long access tubing sequentiallyseparated by air (such that the molar ratio changes along with thesequence of reactant segments). The access tubes can be driven by amulti-port syringe pump. A each molar ratio, the syringe pump candeliver the exact amount of sample and reagent into the reaction chamberfor heat measurement, as well as sample and buffer into the referencechamber. Buffer segments can also be added between two reactant segmentsin the sequence for purposes of cleaning the chamber or mixer.

With further reference to FIG. 29, the differential temperature of thereactions is measured at 2910. The measurement can be accomplished usinga thermoelectric sensor such as a thermopile. The thermopile can outputa voltage indicative of the differential temperature. The output voltagecan then be adjusted based on the baseline in device output measured at2904.

The differential temperature can then be used to determine a thermalpower related to the reaction at 2912. The thermal power difference ΔPcan be calculated as:

$\begin{matrix}{{\Delta\; P} = \frac{\Delta\; U}{S}} & (1)\end{matrix}$where ΔU is the output from the thermoelectric sensor and S is thethermoelectric sensitivity, i.e., the output electrical voltagegenerated by unit differential thermal power.

The differential thermal power can then be used to calculate thethermodynamic reaction parameters at 2914. In general, a biochemicalreaction between a sample molecule M and a binding reagent X can berepresented as:n ₁ X+n ₂ M→MX+ΔH  (2)where the reaction results in the product MX accompanied by a change ofenthalpy ΔH. In ITC, the binding reagent X is titrated, i.e.,successively added in known aliquots, into the sample, while thereaction heat is measured. The reaction heat is measured. The reactionheat is used to calculate the thermodynamic properties of the reaction,including the equilibrium binding constant KB=[MX]/[X][M] (where [⋅]denotes the equilibrium concentration of the species), stoichiometryN=n₁/n₂, and molar enthalpy change ΔH. In particular, the reaction heatcan be calculated based on the differential thermal power. Thebiochemical reaction heat can be expressed as:

$\begin{matrix}{Q = {\frac{{NM}_{t}\Delta\;{HV}_{0}}{2}\left\lbrack {1 + \frac{r}{N} + \frac{1}{{NK}_{B}M_{t}} - \sqrt{\left( {1 + \frac{r}{N} + \frac{1}{{NK}_{B}M_{t}}} \right)^{2} - \frac{4r}{N}}} \right\rbrack}} & (3)\end{matrix}$where Q is the biochemical reaction heat evolved at a molar ratior=X_(t)/M_(t), V₀ is the active volume for the reaction, M_(t) is thetotal concentration of the sample, free plus bound, in the reaction cellof volume V₀, and X_(t) is the total concentration of the reagent thatis titrated into the sample solution.

In order to calculate the thermodynamic reaction parameters, an integralof the differential thermal power is computed. The resulting value isused as the biochemical reaction heat. A number of data points can begathered based on the voltage measurements from a number of trials usingdifferent molar ratios. The resulting data can then be fitted toEquation (3) in order to calculate the thermodynamic reactionparameters. Fitting can be accomplished using fitting methods as knownin the art for its intended purpose.

FIG. 4 is a schematic representation of the exploded view of amicrodevice that is particularly suited for the ITC. The microdevice(400) includes a sample chamber (410) and a reference chamber (420),both situated on the thin film substrate (450) which includes athermopile (470) for measuring the temperature differential of thesample chamber and the reference chamber during a heat scan. Tofacilitate mixing of the first and second substances (FIGS. 4A and 4B),the microdevice further includes introduction channels (430, 440) foreach of the sample and reference chambers (410, 420). Each of theintroduction channels has two inlets (431, 432; 441, 442). Each of theintroduction channels can be configured to provide passive chaoticmixing for a solution flowing through the channel. For example, asschematically shown in FIGS. 4a and 4b , the introduction channels (430,440) can include a portion having a serpentine shape. Moreover, theintroduction channels (430, 440) can includes internal ridges (434, 444)sufficient for creating turbulence in the solution flowing throughchannels. For example, as shown in the inlet of FIG. 4A, theintroduction channels can include herringbone-shaped ridges in theceiling.

The disclosed microdevice and methods of fabrication and use thereof arefurther illustrated in the examples below, which should not beconsidered as limiting the scope of the disclosed subject matter in anyway.

Example 1: Fabrication of Microdevice

This example illustrates a procedure to fabricate the microdevice, whichsubstantially follows the outlined procedure described above inconnection with FIGS. 2A-2E. In particular, a 6-μm thick polyimide filmwas spin-coated on a silicon wafer (precoated with silicon dioxide). TheTMAH etching into the backside of the wafer in the areas that correspondto the calorimetric chambers created an approximately 50 μm-thickresidual wafer layer. After the curing of the polyimide, a chromium/goldthin film (5/200 μm) was deposited by thermal evaporation onto thepolyimide layer. A second layer of polyimide was then coated on themicroheaters and temperature sensors. Subsequently, Sb and Bi thin films(0.5 and 1.2 μm) were thermally evaporated and patterned using astandard lift-off process to form a 50-junction thermopile using astandard lift-off process. A layer containing polyimide-PDMS mixture wasfurther coated on the thermopile. The chamber housing structure wasfabricated from PDMS using micromolding techniques on top of the thinfilm substrate, thereby forming the calorimetric chambers thecalorimetric chambers each of cylindrical shape and 1 μL in volume(diameter: 2.5 mm and height: 200 μm), with a center-to-centerseparation of 4 mm. Xenon difluoride (XeF₂) gas-phase isotropic etchingwas used to remove the residual silicon layer on the wafer substratefrom the backside of the thin film substrate. The integrated resistivemicroheaters each had a nominal resistance of 40Ω and the temperaturesensors each had a nominal resistance of 55Ω. Shown in FIGS. 5A-5D arethe images of the PDMS housing structure and solid thermal substrate, aswell as micrographs of the thermopile, integrated microheater andtemperature sensor embedded in the thin film substrate.

Example 2: Calorimetric Measurement

In this example, the microdevice as fabricated according to Example 1was calibrated and used to measure thermodynamic properties of certainbiomolecules, e.g., thermodynamics of the unfolding of a protein.

A. Principle

DSC can measure differential heat capacity, i.e., the heat capacitydifference between a sample and a reference material, as a function oftemperature. When the sample and reference materials are subjected toidentical temperature scanning, i.e., their temperatures are varied at apredetermined rate within a range of interest, the thermally inducedactivity of the sample molecules, which is either exothermic orendothermic, can cause a small temperature difference between the sampleand reference materials (i.e., differential temperature or temperaturedifferential). This differential temperature can be detected to reflectthe differential powerΔP=P _(s) −P _(r)  (4)where P_(s) and P_(r) are the thermal power generated in the sample andreference materials, respectively. Therefore the differential heatcapacityΔC _(p) =C _(ps) −C _(pr)  (5)where C_(ps) and C_(pr) are, respectively, the heat capacities of thesample and reference materials, can be determined as:

$\begin{matrix}{{\Delta\; C_{p}} = {\frac{\Delta\; P}{\overset{.}{T}} = \frac{\Delta\; U}{S\overset{.}{T}}}} & (6)\end{matrix}$where {dot over (T)} is the time rate of the controlled temperature ofsample and reference materials, U is the output from the thermoelectricsensor that is employed to detect the differential temperature, and S isthe device's sensitivity, i.e., the output electrical voltage generatedby unit differential thermal power. Therefore, interpretation of thedifferential heat capacity can lead to determination of the fundamentalthermodynamic properties of the sample material.B. Device Calibration

In order to measure the temperature differential between the twochambers, the thermopile need be first calibrated such that the voltagegenerated by the thermopile can be readily convert to temperaturedifferential. As illustrated in FIG. 6A, to calibrate the MEMS DSCdevice, the on-chip microheaters were driven by a DC power supply(Agilent E3631A) and generated a constant differential heating power inthe calorimetric chambers, while the temperature sensors wereinterrogated by a digital multimeter (Agilent 34410A) to monitor thetemperatures of the calorimetric chambers. The thermopile outputvoltage, proportional to the differential temperature between thechambers, was measured by a nanovoltmeter (Agilent 34420A). Thetemperature control of the MEMS DSC device and thermoelectricmeasurements were automated using a personal computer via a LabVIEW-based program.

A packaged MEMS DSC device (100) was housed in a custom-built,temperature-controlled thermal enclosure (200) consisting of multiplemetal enclosures surrounding a metal stage on which the device wasplaced (FIG. 6B). This provided temperature scanning of the sample andreference solutions, as well as thermal isolation of the device packagefrom the environment to minimize measurement noise. Multiple Peltierdevices (Melcor UT15-12-40-F2) were located underneath the device stage,and by a power supply (Agilent E3631A), to add heat to or remove heatfrom the device. The temperature of the sample and reference chamberswas controlled in closed loop by adjusting the voltage applied to thePeltier devices according to the feedback from the on-chip temperaturesensors based on, for example, a proportional-integral-derivative (PID)algorithm.

During device calibration, the sample and reference chambers were bothfilled with 0.1 M Glycine-HCl buffer (pH 2.5), which was the bufferlater used for protein unfolding measurements. A known, constantdifferential power was created by activating the microheater below thesample chamber while leaving the microheater underneath the referencechamber turned off. The temperature sensors were used to measure thetemperatures of the thermopile's hot and cold junctions. The deviceoutput, i.e., the thermopile output voltage, was measured as a functionof time to obtain the steady-state and transient responses to thedifferential heating power.

The sensitivity of the thermopile integrated in the MEMS DSC device wascalibrated at varying temperature difference between the hot and coldjunctions, generated by on-chip heating (using the microheaterunderneath the sample chamber). The thermopile differential voltageexhibited a highly linear relationship with temperature difference (FIG.7), showing a total thermoelectric sensitivity of 6.3 mV/° C. for the50-junction thermopile. A Seebeck coefficient of 125 μV/K for each Sb—Bithermocouple was obtained. In addition, the steady-state response of theMEMS DSC device was calibrated to varying differential power andobserved again a highly linear relationship, yielding a nearly constantresponsivity of S=4.0 mV/mW (FIG. 8). A root-mean-square (RMS) noise ofapproximately 40 nV in the device output was also observed, which wasused to determine a baseline noise in the differential power. Thiscorresponded to a detection limit of approximately 10 nW in differentialthermal power measurement.

To characterize the transient response of the MEMS DSC device, a stepdifferential power of 130 μW was initially applied to the calorimetricchambers and then turned off once the device output reached itsequilibrium. The corresponding output voltage from the thermopile (FIG.9) was found to exponentially grow with time upon the application of thedifferential power, while decay exponentially upon the removal of thedifferential power. The thermal time constant was approximately 2.0 s,calculated by fitting the experimental data to first-order exponentialgrowth and decay functions.

C. Calorimetric Measurements

DSC measurements of biomolecules were performed using the calibratedmicrodevice, whose sample chamber and reference chamber wererespectively filled with biological sample and buffer solutions, scannedin a range of temperature of interest. The temperature sensors were usedto monitor the temperatures of calorimetric chambers while the deviceoutput was obtained in real time to compute the biomolecular thermalpower. Before DSC measurements, the baseline in device output, i.e., thethermopile output voltage in the absence of a differential power input,during temperature scanning was measured with both calorimetric chambersfilled with buffer solutions. Biological sample and buffer solutionswere degassed with a vacuum chamber built in-house, metered withmicropipettes, and introduced by a syringe pump (New Era Pump Systems,NE 1000).

The calibrated MEMS DSC device was employed to characterize proteinunfolding, a common type of biomolecular conformational transition. Forthis purpose, the thermal enclosure provided temperature scanning of theMEMS DSC device at time rates as high as 6° C./min in the range of10-90° C. with power consumption lower than 25 W. Using lysozymeprepared in 0.1 M Glycine-HCl buffer (pH 2.5) for purposes ofdemonstration, the device output was monitored while the sample andreference chambers, respectively filled with lysozyme and buffer, werescanned in a temperature range of 25-75° C. at a constant rate of 5°C./min.

The thermopile output voltage as a function of temperature, corrected bybaseline subtraction, was measured at varying protein concentrationsranging from 1 to 20 mg/mL (FIG. 10). It was observed that the deviceoutput exhibited a concentration-dependent minimum within a certaintemperature range, reflecting the endothermic nature of proteinunfolding processes. Notably, the unfolding of lysozyme was detectableat 1 mg/mL, representing a significant improvement over the previouslyreported MEMS DSC device.

Furthermore, the differential heat capacity between the chambers wascomputed from the differential voltage measurement (FIG. 10) usingcalibrated device sensitivity (4.0 mV/mW), allowing the thermodynamicproperties of lysozyme to be obtained during its unfolding process, suchas partial specific heat capacity (c) (FIG. 11A), the total change ofmolar enthalpy (i.e., enthalpy per mole of lysozyme) (ΔH), and meltingtemperature (T_(m), defined as the temperature at which the change ofmolar enthalpy achieves 50% of ΔH) (FIG. 11B). Despite the amplitudedifference of device output at various protein concentrations, they allyielded consistent estimates of the thermodynamic properties associatedwith the protein unfolding process. In particular, the profile shape ofc was generally not influenced by protein concentration, and ΔH wasconsistently determined to be approximately 450 kJ/mol with acorresponding melting temperature T_(m) of approximately 55° C. Theseresults agree well with published data, which are typically in the rangeΔH=377-439 kJ/mol and T_(m)=55-58.9° C. for lysozyme, demonstrating thepotential utility of the MEMS DSC device disclosed herein forbiomolecular characterization with significantly reduced sampleconsumption at practically relevant protein concentrations.

The effects of the temperature scanning rate on DSC measurements werealso investigated. Using 20 mg/mL lysozyme prepared in 0.1 M Glycine-HClbuffer (pH 2.5) for example, the unfolding of lysozyme at temperaturescanning rates were varied from 1-6° C./min. The thermopile outputvoltage (again corrected by baseline subtraction) (FIG. 12A) exhibited aconsistent dip in the same temperature range for protein unfolding asindicated above, with an amplitude increasing with the temperaturescanning rate. This is consistent with a larger heat flux resulting in ahigher endothermic power through phase transformations.

These data were then used to compute the change of molar enthalpy (FIG.12B). Although a slight shift in the device output could be observed(FIG. 12A) as temperature scanning rate increased, the thermodynamicproperties associated with the protein unfolding process were foundgenerally consistent, with a standard variation in ΔH of approximately50 kJ/mol (i.e., ±5% of the mean value of ΔH) and a standard variationin T_(m) of less than 1° C. (FIG. 12B). Notably, for temperaturescanning at 1-5° C./min, the T_(m) values were almost the same. Thisdemonstrates the measurement consistency using the MEMS DSC device ofthe disclosed subject matter, and indicates that a temperature scanningrate as high as 5° C./min is adequate for the measurement of lysozymeunfolding.

Example 3: AC-DSC Measurement

This Example illustrates the method of carrying out a AC-DSCmeasurement, as described above based on a microdevice presentlydisclosed. This MEMS AC-DSC approach can potentially enable measurementsof low-abundance biomolecules with improved accuracy, as demonstrated bythe application of the device to AC-DSC measurements of the unfolding oflysozyme.

A. Principle

AC-DSC can monitor the differential heat capacity, i.e., the heatcapacity difference between a sample and a reference material, byvarying the materials' temperatures at a specified constant rate via athermally isolated enclosure equipped with temperature controlfunctionalities, superimposed with a temporally periodic variation viaidentical AC modulation heating applied to the sample and reference(FIG. 3). The differential heat capacity can be obtained by themeasurement of the differential temperature, i.e., the temperaturedifference between the sample and reference materials.

B. Fabrication of the Microdevice, System Setup and Calibration

The AC-DSC measurement was carried out using a microdevice schematicallydepicted in FIG. 1A-1C and fabricated according to the proceduredescribed in Example 1. While other device parameters, including thedimension and volume of the chambers, thickness of polyimideparaphragms, and characteristics of the microheaters and temperaturesensors, are largely the same as those of the microdevice described inExample 1, the Sb—Bi thermopile used in this Example includes 100junctions rather than 50 junctions in Example 1.

The DSC measurement system was configured similarly to that of Example2. The microdevice was also placed in a thermal enclosure builtin-house. The temperature of the sample stage in the thermal enclosurewas controlled in closed-loop via a proportional-integral-derivative(PID) algorithm implemented by a commercial temperature controller(Lakeshore 331). The on-chip microheaters driven by a DC power supply(Agilent E3631A) were used to generate a constant differential powerinput, while for modulated heating, a square-wave AC voltage generatedby a waveform generator (Agilent 33220A) was applied (FIG. 13). Thetemperature sensors were used to detect the real-time temperature insideeach of the calorimetric chambers by a digital multimeter (Agilent34410A). During device calibration, the thermopile output voltage wasmeasured by a nanovoltmeter (Agilent 34420A), while during AC-DSCmeasurement, the amplitude and phase of thermopile voltage were measuredby a lock-in amplifier (Stanford Research Systems SR830) referenced bythe same AC modulation square wave from the waveform generator. TheAC-DSC measurement was fully automated through a Lab VIEW program.

The methods for calibrating the DC performance of the MEMS device weresubstantially the same as described in Example 2. The baseline in deviceoutput, i.e., the thermopile voltage with no differential power inputduring temperature scanning, was measured with both chambers filled withbuffer solutions. During calibration of the device's modulationfrequency dependence and AC-DSC measurements, the sample chamber wasfilled with a biological sample solution while the reference chamber wasfilled with the buffer solution. Biological sample and buffer solutionswere degassed with a vacuum pump built in-house and then introduced intothe device's calorimetric chambers with micropipettes.

The thermopile in the MEMS device was first calibrated, and the resultsshowed that 100-junction thermopile had a sensitivity of 13.0 mV/° C.(FIG. 14), corresponding to a Seebeck coefficient of (per Sb—Bithermoelectric junction) of approximately 130 μV/° C. The steady-stateresponse of the device to a constant differential power was thenmeasured, and exhibited a highly linear relationship with a DCresponsivity of 8.0 mV/mW (FIG. 15). These results were consistent withcalibration results from the 50-junction Sb—Bi thermopile in Example 2.Additionally, the transient response of the device was determined. Thecalorimetric chambers, both of which were filled either with air or with0.1 M Glycine-HCl buffer (pH 2.5), were subjected to a step differentialpower (0.32 or 1.30 mW). Results from these measurements are shown inFIG. 16. The dependence of the thermopile voltage on time can berepresented by a first order exponential increase. The thermal timeconstant thus obtained was 0.8 s when the chambers were filled with air,and 2.0 s when they were filled with buffer solution. These values wereindependent of the applied differential power, and were smaller thanconventional AC calorimetric measurements.

Further, the modulation frequency dependence of the device response tothe applied differential power was investigated. To better simulate theapplication for AC-DSC measurement of protein unfolding process, thesample chamber was filled with lysozyme (20 mg/mL, prepared in 0.1 MGlycine-HCl, pH 2.5) as a sample, while the reference chamber was filledwith Glycine-HCl buffer. The chambers were maintained at a constanttemperature (25, 35, or 45° C.), and subjected to AC heating (voltageamplitude: 1 V). The dependence of the thermopile voltage amplitude onthe modulation frequency, corrected by baseline subtraction, is shown inFIG. 17. It can be seen that the thermopile voltage increased withtemperature at almost all modulation frequencies, which can be explainedby the temperature-dependence of the protein's heat capacity. Also, thedevice output (and hence sensitivity) appear greatest in a modulationfrequency range of 0.5 to 20 Hz (FIG. 17), suggesting a reduced heatloss to the ambient by choice of modulation frequency. Therefore,modulation frequencies in this range were used below in calorimetricmeasurements of protein unfolding processes, as further described below.

C. AC-DSC Measurements

The MEMS AC-DSC device calibrated above was used to measure the thermalbehavior of protein unfolding. Using lysozyme at differentconcentrations (10 and 20 mg/mL, prepared in 0.1 M Glycine-HCl buffer,pH 2.5) for example, the temperature of the calorimetric chambers wasvaried from 25 to 82° C. at a rate of 5° C./min in combination with ACmodulation via a heating voltage amplitude of 3.5 Vat a constantfrequency (1, 5, or 10 Hz). The periodic temperature variation resultingfrom the AC modulation heating had an amplitude of approximately 0.2° C.

The measured thermopile voltage amplitude (FIG. 18A), again corrected bybaseline subtraction, showed a concentration-dependent dip during theunfolding process, consistent with the endothermic nature of proteinunfolding. In addition, despite differences in thermopile voltageamplitude for different lysozyme concentrations, the phase of thethermopile voltage (FIG. 18B) had identical changes throughout theunfolding process, which remained unchanged in the native and unfoldedstates when a two-state protein denaturation model was adopted.Furthermore, both the amplitude and phase changes of the thermopilevoltage exhibited clear shifts with the modulation frequency, whichcould be attributed to the unsynchronized thermal response of the deviceto AC heating. However, at a fixed protein concentration, the profilesof the thermopile voltage amplitude and phase changes had virtually thesame shape at different modulation frequencies, showing the suitabilityof the frequency choice for the MEMS-based AC-DSC measurements.

The apparent melting temperature (T_(m)) of lysozyme during an unfoldingprocess, i.e., the temperature at which the phase change of deviceoutput reaches its peak, was found to be in the range of 55-58° C. (FIG.18), depending on the modulation frequency. Meanwhile, the specific heatcapacity (c) of the protein as a function of temperature can be computedfrom the thermopile voltage amplitude (FIG. 19). It be seen thatalthough there again existed a slight shift in c throughout theunfolding process induced by modulation frequency, the profile shape ofc was not influenced by the modulation frequency. Moreover, at eachmodulation frequency, the calculated value of c does not differsignificantly at different protein concentrations (FIG. 19), showingthat the AC-DSC measurements were accurate. There was also a differencein the specific heat capacity (Δc) between the protein's native andunfolded states, which was calculated consistently to be 3.0 kJ/mol·Kregardless of the modulation frequency. These results are consistentwith established results from DC-DSC characterization. Compared withDC-DSC measurements in the same MEMS device without using temperaturemodulation (FIG. 20), AC-DSC can offer much reduced noise levels andimproved measurement accuracies, and therefore holds the potential toenable characterization of biomolecular interactions at lowconcentrations

Example 4: MEMS-Based Isothermal Titration Calorimetry

This Example illustrates the method of performing isothermal titrationcalorimetric measurement, as described above based on a microdevicedisclosed herein.

A. Principle

Consider a solution-phase biochemical reaction n₁A+n₂B↔C+ΔH, where A andB are reactants (e.g., a ligand and a sample, respectively) and C is aproduct. The reaction is accompanied by a change of enthalpy ΔH. In ITC,the ligand can be titrated, or successively added in known aliquots,into the sample, while the reaction heat is measured. This data can thenused to determine the thermodynamic properties of the reaction,including the equilibrium binding constant K_(B)=[C]/[A][B] (the squarebrackets denote the equilibrium concentration of the species),stoichiometry N=n₁/n₂, and enthalpy change (ΔH).

B. Device Setup and Calibration

The MEMS-ITC device as schematically shown in FIG. 4A-4B was used.Briefly, the device integrates two identical polydimethylsiloxane (PDMS)microchambers each (1 μL) situated on a freestanding polyimide thin filmsubstrate and surrounded by air cavities for thermal isolation. Thechambers are integrated with an antimony-bismuth (Sb—Bi) thermopile andconnected to the inlets through an introduction channel which includes apassive chaotic mixer having herringbone-shaped ridges in the ceiling ofa serpentine channel to generate a chaotic flow pattern that inducesmixing of the incoming liquid streams. Some of the features of theMEMS-ITC device used were shown in FIG. 21A-21B. For ITC measurements,the two reactants, herein referred to as the ligand and sample forpurpose of illustration, were introduced into the device and first mixedin the introduction channel, and then enter the sample calorimetricchamber, where the reaction is completed. In the meantime, the sampleand pure buffer (devoid of the ligand) were also introduced into thedevice, becoming mixed before entering the reference calorimetricchamber. The differential temperature between the chambers were measuredusing the integrated thermopile, and was used to determine the thermalpower from the reaction, from which the thermodynamic reactionparameters were calculated. The device was placed in a low-noise,temperature-controlled thermal enclosure (FIG. 22) where the thermopileoutput was measured. The sample and ligand were introduced using syringepumps. Calibration experiments indicated that the device had a thermaltime constant of 1.5 s with a linear steady-state thermal response(responsivity: 4.9 mV/mW) (FIG. 23A-23B).

C. ITC Measurement

The device was used for ITC measurements of a model reaction systemconsisting of 18-C-6 and BaCl₂. The time-resolved device outputexhibited a reaction-specific spike (FIG. 24) upon introduction of 5 mMBaCl₂ and 4 mM 18-C-6 (each 0.5 μL) with no appreciable delay,indicating full mixing of the reactants. Using titrations with the molarratio (BaCl₂/18-C-6) varying from 0.1 to 2 ((1) 0.1, (2) 0.4, (3) 0.8,(4) 1.0, (5) 1.2, (6) 1.6, (7) 2.0), the baseline-subtracted deviceoutput demonstrated spikes consistent with the titration reactions, andallowed construction of a binding isotherm (FIG. 25A-B). ITCmeasurements were performed at 23 and 35° C. (FIG. 26), and theresulting isotherms were used to compute K_(B) and ΔH, which decreasewith temperature (See Table 1 below). These results demonstrated thatthe MEMS-ITC device as disclosed affords detectable sampleconcentrations approaching those of conventional instruments (ca. 1 mM)with roughly three orders of magnitude reduction in volume.

TABLE 1 Temperature-dependent thermodynamic properties: thestoichiometry (N), binding affinity (KB) and enthalpy change (H), of thebinding of 18-C-6 and BaCl2 at two temperatures Temperature (° C.) NK_(B)(M⁻¹) ΔH(kJ/mol) 23 1.0 6.0 × 10⁻³ 30.0 35 1.05 2.8 × 10⁻³ 27.8

Example 5: MEMS-Based Isothermal Calorimetry

Chaotic mixers and calorimetric chambers were fabricated in a singlesheet using PDMS replica technique based on multiple-layered SU-8molding. The microfabricated device integrated a 50-junction Sb—Bithermopile and two 0.75 μL calorimetric chambers with a center-to-centerseparation of 4 m. The calorimetric chambers had a cylindrical shapewith a height of 150 μm and a diameter of 2.5 mm. The chaotic mixerswere serpentine microchannels (width: 200 μm, height: 150 μm, length:approximately 15 mm) with herringbone-shaped ridges on the ceiling witheach having a width of 40 μm, height of 50 μm, an orientation angle of60° to the channel sidewall, and an edge-to-edge distance betweenadjacent ridges of 30 μm. The nominal resistances of the integratedresistive microheaters and temperature sensors were 40Ω and 55Ω,respectively.

To test the MEMS-IT device, a thermal enclosure was custom-built tohouse the device to shield the thermal disturbance from ambient, as wellas provide uniform temperature control to the solutions loaded in thedevice. The thermal enclosure was improved with additional thermalisolation by suspending the sample stage from the base, vibrationisolation by enhanced base mass and rubber buffering layer, andmultiple-ports microfluidic feedthrough to the device. The temperaturecontrol of the thermal enclosure was implemented by a commercialtemperature controller (Lakeshore Model 331). The device was firstpackaged with electrical interconnection wires and fluidicinterconnection tubes before it was situated on the sample stage insidethe thermal enclosure.

The on-chip microheaters, used for device calibration, were driven by aDC power supply (Agilent E3631A) and generated a constant differentialheating power in the calorimetric chambers. The on-chip temperaturesensors, used for in-situ temperature monitoring of the calorimetricchambers, were interrogated by a digital multimeter (Agilent 3410A). Thethermopile output voltage, which is proportional to the differentialtemperature between the chambers, was measured by a nanovoltmeter(Agilent 34420A). The temperature monitoring of the calorimetricchambers and thermoelectric measurements were automated using a personalcomputer via a Lab VIEW-based program. The biological sample and buffersolutions were degassed with a vacuum chamber built in-house, meteredintroduced into the MEMS-ITC device using a multiple-injections syringepump (KD Scientific, KDS 220).

The device was first calibrated by measuring its steady-state andtransient response to differential power generated by on-chipmicroheaters. Before ITC measurements, the baseline in device output,i.e., the thermopile output voltage in the absence of reaction, wasmeasured with introduction of sample and buffer solutions to bothcalorimetric chambers. During ITC measurements, the thermal enclosureprovided a controlled reaction temperature while the thermopile output,indicative of the differential bio-thermal power, was detected in realtime, as well as the integrated micro-temperature sensor to monitor thetemperatures of the calorimetric chambers. The volume of ligand andsample was fixed at 0.5 μL for each injection, while the molar ratio wasadjusted by changing the concentration of ligand to be injected. Thebaseline in device output was always subtracted from the measurementsignal for determination of thermodynamic properties of biomolecules.

The thermal time constant of the MEMS-ITC device was calibrated byapplying a step differential power of 90 μW initially and then turned itoff once the device output reached its equilibrium. The device outputvoltage was found to fit the first-order exponential growth and decayfunctions upon the application and removal of the differential power,respectively, from which the thermal time constant was determined to beapproximately 1.5 s. In addition, the steady-state response of thedevice was calibrated to varying differential power, and a linearrelationship showing a constant thermoelectric sensitivity of S=4.9mV/mW was observed. The device's sensitivity was also calibrated atcontroller temperatures (provided by the thermal enclosure) from 20° C.to 45° C., and it was found that it remained almost unchanged with arelative standard deviation of less than 3%.

The baseline stability and detection specificity was then tested using astandard chemical reaction of 18-Crown-6 (18-C-6) and barium chloride(BaCl₂) both prepared in sterile water (all chemicals from SigmaAldrich). Using a flow rate of 50 μL/min, the solutions were injectedinto the calorimetric chambers within 1 s. Using a data acquisition rateof 2 s⁻¹ to monitor the device output in real time, no appreciable delaywas observed after injection, indicating full mixing of the reactants. Acomparison of the time-resolved thermopile voltage upon introduction of4 mM BaCl₂ and 5 mM 18-C-6 (each 0.5 μL) in the reaction chamber, andthe signal upon introduction of sterile water and 5 mM 18-C-6 (also each0.5 μL) is shown in FIG. 30. For both measurements, the referencechamber was injected with sterile water and 5 mM 18-C-6, and a dataacquisition rate of 0.2 s⁻¹ was used due to instrument configuration forlower background noise. The device exhibited a stable baselinethroughout the measurements and a reaction-specific spike attributableto the exothermic nature of the binding between 18-C-6 and BaCl₂. Thereaction completed in approximately 20-30 s, during which anyinterference from solution injection and mixing were generallynegligible.

The MEMS-ITC device was then used to characterize biomolecularinteractions. The BaCl₂-18-C-6 reaction was used to validate the ITCmeasurements. By varying the molar mass ration (MBACl₂/M18-C-6) from 0.1to 2, the baseline-subtracted device output demonstrated spikesconsistent with the titration reactions. The baseline-subtracted deviceoutput is shown in FIG. 31. Rather than measuring the heat evolved withthe addition of several aliquots of BaCl₂ to a single sample of 18-C-6as performed in commercial ITC instruments, the ITC experiment wasperformed at discrete measurements each with a definite concentration ofBaCl₂ (0.5-10 mM) and a fixed concentration of 18-C-6 (5 mM). Eachmeasurement was completed in approximately 5 min.

The thermopile voltage was the used to calculate the bio-thermal powerbased on Equation 1. The bio-thermal power was then used to calculatethe reaction heat by integral of the biothermal power during theprocess.

The binding isotherm of the reaction of 18-C-6 and BaCl₂, as well as thefitted curve, is shown in FIG. 32, with error bars representing thestandard deviation from three measurements a each molar ratio. Note thatfor this specific BaCl2-18-C-6 system, the device affords detectablesample concentrations approaching those of convention instruments (ca. 1mM) with roughly three orders of magnitude reduction in volume.

ITC measurements were performed of the biological reaction of 18-C-6 andBaCl2 at controlled temperatures of 23° C. and 35° C., and the resultingbinding isotherms were used to compute the temperature-dependentthermodynamic properties of N, K_(B), and ΔH. In particular, astemperature increases from 23° C. to 35° C., N slightly increases from1.00 to 1.05, while K_(B) decreases from approximately 6.0×10⁻³ to2.0×10⁻³ M⁻¹ and ΔH decreases from 30.0 o 27.8 kJ/mol, showing a trendof slightly weaker binding with temperature. These properties and theirtemperature dependence obtained by suitable measurements agreereasonably with published data using commercial calorimeters as shown inFIG. 33.

The MEMS-ITC device was further applied for characterization ofbiomolecular interactions, e.g., ligand-protein binding, using ademonstrative system of cytidine 2′-monophosphate (2′CMP) andribonuclease A (RNase A). 2′CMP is known as a strong inhibitor ofsubstrates that bind to the active site of RNase A. Both reagents wereprepared in 50 mM potassium acetate buffer, pH 5.5. Similarly, atvarying molar rations (2′CMP/RNase A) from 0.1 to 2, the device outputexhibited titration-dependent spikes in correspondence to the molarratio as shown in FIG. 34. ITC measurements of 2′CMP-RNase A binding atcontrolled temperatures of 23° C. and 35° C. with error bars from threemeasurements at each molar ration were also performed. In turn, thetemperature-dependent thermodynamic properties associated with thisbiomolecular interaction were determined from fitting the experimentaldata to the described model in Equation 3. The results again agreedreasonably with published data using commercial ITC instruments as shownin FIG. 35. For 2′CMP-RNase A interaction, the reasonably detectableconcentration of RNase A can be as low as 2 mM. These resultsdemonstrate the utility of this MEMS-ITC device for efficientcharacterization of a wide variety of biomolecular interactions.

Example 6: Polymer-Based MEMS Differential Scanning Calorimeter

The polyimide film which was used as a substrate was purchased fromDuPont (Kapton® 50HN, 12.5 μm thick). The fabrication began with thereversible binding of the substrate to a silicon wafer carrier by aspin-coated poly(dimethylsiloxane) (PDMS) adhesive layer (20 μm). Afterfully curing this PDMS adhesion layer, Sb and Bi (40 μm wide, 2 mm long,0.5 and 1 μm thick, respectively) were thermally evaporated andpatterned on the substrate using a standard lift-off process to form a400-junction thermopile, which was then passivated with a spin-coatedpolyimide thin layer (1.5 μm). Subsequently, a chromium/gold thin film(5/150 nm) was deposited and patterned to define the on-chip temperaturesensor, which had a nominal resistance of 55Ω, and then was passivatedwith another intermediate layer of polyimide-PDMS blend which alsoserved as the adhesion layer of the PDMS microfluidic structure. Thefabricated devices were mechanically released from the substrate, andthe PDMS binding layer was peeled off so that the silicon carrier can besaved for reuse. In parallel, serpentine microfluidic channels (width:200 μm, height: 200 μm, length: 25 mm; volume: 1 μL) were fabricated ofPDMS via soft lithography. The released substrate was then bonded to themicrofluidic structure via oxygen plasma (100 W 3s).

The packaged polymer MEMS DSC device was placed in a custom-builtthermal enclosure consisting of a metal enclosure cap surrounding analuminum stage on which the device was placed. The thermal enclosureoffered additional thermal isolation of the DSC devices from ambienttemperature aimed to reduce environmental noise. In addition, theenclosure provided an environment in which the sample and referencesolutions in the device were at a sufficiently uniform temperature,which was scanned at a specified rate. Multiple Peltier devices (MelcorUT15-12-40-F2 were located underneath the device stage to add heat to orremove heat from the device while the temperature was precisely scannedwith a lakeshore temperature controller (Lakeshore model 311). Thetemperature of the sample and reference channels was controlled inclosed loop by adjusting the voltage applied to the Peltier devicesaccording to the feedback from the temperature sensors mounted on themetal stage based on a proportional-integral-derivative algorithm. Theon-chip temperature sensor, calibrated before use, was measured by adigital multimeter (Agilent 34410A), provides in situ temperaturemonitoring. The thermopile output voltage was measured by anano-voltmeter (Agilent 34420A). At any instant during DSC measurements,this allowed the biomolecules in the sample solution to experience auniform temperature, which was accurately obtained by on-chiptemperature sensors. Thus, together with the determination of thedifferential power by the thermopile, the MEMS device in thisexperimental setup could achieve accurate DSC measurements ofbiomolecules.

Lysozyme, used as the targeted sample biomolecule, was purchased fromSigma Aldrich (lyophilized powder, protein ≥90%) and dissolved in 0.1-Mglycine-HCL buffer (pH 2.5). The sample solutions and buffer weredegassed overnight in a vacuum chamber built in-house, metered withMicropipettes, and introduced by a syringe pump (New Era Pump Systems,Inc., NE-1000) before the DSC measurements.

Before the polymer DSC device was used to characterize targetbiomolecules, liquids with well-established heat capacities were chosento calibrate the MEMS DSC device. Water and glycerol were used incalibration for their relatively high boiling temperatures whileenvironmental disturbances were minimized by placing the device insideof the thermal enclosure. The thermoelectric and resistive measurementswere automated through a Lab VIEW-based program. After calibration, thedevice was thoroughly washed with buffer and deionized water.

Before DSC measurements, the baseline, i.e., the thermopile outputvoltage in the absence of a differential power input, during temperaturescanning in the range of interest was recorded with both calorimetricchannels filled with buffer solutions. After which, characterizations ofbiomolecules were performed with the calorimetric channels, respectivelyfilled with a biological sample and a buffer solutions, and scanned inthe same range with pre-specified rate. The temperature sensors wereused to monitor the temperatures of calorimetric channels, while thedevice output was obtained in real time to compute the biomolecularthermal power.

The polymer DSC device was calibrated to determine the responsivity ofthe device output. The device temperature was first scanned with bothcalorimetric channels filled with air to account for the effect ofcalorimetric channel volume mismatches. Then, water and glycerol weresuccessively introduced into the sample channel, while the referencechannel remained filled with air. The heat capacities of all materialswere obtained from the literature. The device responsivity wasdetermined using:

$\begin{matrix}{{\Delta\; C_{p}} = {\frac{\Delta\; P}{\overset{.}{T}} = \frac{\Delta\; U}{S\overset{.}{T}}}} & (7)\end{matrix}$

where ΔU is the measured thermopile output voltage, {dot over (T)} isthe constant rate (scanning rate) at which the sample and referencetemperature are varied in the range of interest, and S the deviceresponsivity determined via device calibration, i.e., the thermopileoutput voltage per unit differential power. The device responsivity wasused later for determining the sample heat capacity. All calibrationexperiments were thermally scanned with a rate of 3 K/min to beconsistent with reported measurements in literature. The deviceresponsivity was then determined to be 4.78 mV/mW. As shown in FIG. 39,when the device responsivity, S, and scanning rate, {dot over (T)}, aresubstituted back into Equation 7, the experimentally determined changein heat capacity between glycerol and water as a function of temperatureagree with the calculation using data reported in the literature. Wealso observed an experimental output voltage noise level ofapproximately 100 nV in the DSC detection system, which correlated to adifferential power noise of 21 nW.

Numerical analysis of heat transfer in the polymeric MEMS DSC device wasalso performed to assess the temperature uniformity and verify theresponsivity of the device. Using COMSOL (version 4.4), thethree-dimensional model which includes water filled polymericmicrostructures, thermopile junctions, Kapton substrate and all thepassivation layers in between, accounts for heat conduction inside thedevice and convection from the device's outer surfaces to the ambient.The model assumes steady state transfer at each temperature during thetemperature ramping process, which occurs at a low rate (5 K/min).

Natural convection inside the microchannels is neglected in thesimulation. Natural convection in the water can be characterized by theRayleigh number,

${Ra}_{H} = \frac{g\;{\beta\Delta T}_{\max}H^{3}}{\alpha\; v}$where H is the height of the channel, α thermal diffusivity, β thecoefficient of volumetric thermal expansion, v kinematic viscosity ofwater, g the gravitational acceleration, and ΔT_(max) the maximumtemperature difference between ambient and device layer of interest. Forthe condition where a constant temperature boundary condition isapplied, natural convection can be considered negligible if Ra_(H)<1708.For the geometry and operating conditions (ΔT_(max) up to 70° C.) of thedevice, it is estimated that Ra_(H)˜1.6E-06. It follows that the neglectof the natural convection in the channel is justified.

The model uses the following boundary conditions. Neglecting the thermalcontact resistance at the interface of the substrate and the underlyingPeltier heater, the back side of the Kapton substrate is prescribed atthe temperature of the heater surface. The convection coefficient h,representing the natural convection from the outer surfaces of thedevice to the ambient, is obtained using a correlation in the Nusseltnumber, which is defined by Nu=hL/k and represents the relativesignificance of convection to conduction. Here, k is the thermalconductivity of the air, L the characteristic length (height of thePDMS) For natural convection above a flat isothermal plate, the Nusseltnumber is given by the correlation Nu=0.59*Ra_(air) ^(0.25), whereRa_(air) is the Rayleigh number for air.

A power generation of 3 mW is applied to the entire sample channel torepresent the biological heat generation during the experiments. Thethermal conductivity, specific heat capacities and mass densities of thefluids are temperature dependent and are accounted for in the COMSOLsimulations. The temperature distribution within the microfluidicstructure is shown in FIG. 40. Temperature distribution of the devicewhen substrate is prescribed with temperature: (A) 298 K (B) 318 K (C)338 K (D) 368 K. h is estimated as 5.18, 9.89, 11.57, 13.06 W m⁻² K⁻¹accordingly. The thermal conductivities of antimony, bismuth, Kapton,PDMS are given nominal values of 24.4, 7.97, 0.12, 0.15 W m⁻¹ K⁻¹,respectively. It can be shown that the maximum temperature differencebetween the sample and reference channel is approximately 6° C. when thesubstrate temperature was prescribed to 95° C., indicating the excellentthermal insulation of the polymer DSC device.

To estimate the device responsivity, the average temperature differenceacross thermopile hot/cold junctions is obtained first from simulationresults (varies between 0.6 to 1° C.). Equation 7 is then used to obtainthe device responsivity to be 4.09 mV/mW, which is consistent with theexperimentally obtained value of 4.78 mV/mW, with the deviationattributable to variations in sensor geometries and material propertiesthat are commonly process-dependent. This device responsivity has beenfound to differ by no more than 15% at different substrate temperaturesranging from 298 to 368 K.

To characterize the device time response of the MEMS DSC device, aconstant differential power was initially applied to the calorimetricchannels until the device output reached its equilibrium. Thecorresponding output voltage from the thermopile (FIG. 41) was found toexponentially grow with time upon the application of the differentialpower while decay exponentially upon the removal of the differentialpower. The thermal time constant was approximately 2.6 s, calculated byfitting the experimental data to first-order exponential growth anddecay functions.

The calibrated polymer MEMS DSC device was then exploited tocharacterize protein unfolding. Glycine-HCl buffer (0.1 M, pH 2.5) wasfilled in both sample and reference calorimetric channels while thedevice was scanned at a constant rate of 5 K/min. After the scan wascompleted, the device was allowed to cool to room temperature and asecond experiment under identical conditions was performed to test thestability of the baseline. There was minimal fluctuation between the twobaselines. Notably, a non-zero slope was apparent at elevatedtemperatures, possibly as a result of the volumetric mismatch betweenthe reference and sample channels.

Following the measurement of the baseline, lysozyme in 0.1 M Glycine-HCLbuffer (pH 2.5) was introduced into the device sample channel while thereference channel remained filled only with buffer. The characterizationof the unfolding of lysozyme was carried out with identical experimentalconditions used in the baseline determination. The thermopiledifferential voltage as a function of temperature, corrected by baselinesubtraction, was measured at varying concentrations ranging from 1 to 20mg/mL (FIG. 42). The device output exhibited an endothermicthermodynamic profile within the 25-75° C. temperature range at allprotein concentrations. Notably, the unfolding of lysozyme wasdetectable at 1 mg/mL. Furthermore, the differential heat capacity as afunction of temperature and the calibrated device sensitivity can becalculated from the device voltage output by Equation 7. The sample heatcapacity as a function of temperature was determined by:

$\begin{matrix}{C_{sample} = {{C_{buffer}\left( \frac{v_{sample}}{v_{buffer}} \right)} + \left( \frac{\Delta\; C_{p}}{m_{sample}} \right)}} & (8)\end{matrix}$where v_(sample) and v_(buffer) are the partial specific volumes of thesample and the buffer respectively, m_(sample) is the mass of thebiomolecule in the sample channel, and c_(buffer) is the partialspecific heat capacity of the buffer. The interpretation of thefundamental thermodynamic properties, such as the total enthalpy changeper mole of lysozome (ΔH) and melting temperature (T_(m) defined as thetemperature at which the enthalpy change achieves 50% of ΔH) associatedwith this conformational transition can then be determined.

The enthalpy change was determined by:

$\begin{matrix}{{\Delta\;{H(T)}} = {\overset{T_{1}}{\int\limits_{T_{0}}}{{C_{sample}(T)}{dT}}}} & (9)\end{matrix}$

from all protect concentrations except 1 mg/mL, which was excluded fromthe calculations due to the high noise in the thermopile output. Asshown in FIG. 43, ΔH=421 kJ/mol obtained consistently, with acorresponding melting temperature T_(m)=54.71° C. These results agreewith the published data, and thus demonstrate the capability of thepolymer MEMS DSC device for highly sensitive detection of biomolecularinteractions.

The description herein merely illustrates the principles of thedisclosed subject matter. Various modifications and alterations to thedescribed embodiments will be apparent to those skilled in the art inview of the teachings herein. Further, it should be noted that thelanguage used herein has been principally selected for readability andinstructional purposes, and can not have been selected to delineate orcircumscribe the inventive subject matter. Accordingly, the disclosureherein is intended to be illustrative, but not limiting, of the scope ofthe disclosed subject matter.

The invention claimed is:
 1. A microdevice for calorimetric measurement,comprising a first thermally isolated microchamber; a second thermallyisolated microchamber; a thin film substrate formed on a polymericlayer; wherein the first thermally isolated microchamber and the secondthermally isolated microchamber are identical in volume andconfiguration, and arranged side by side, each supported on the thinfilm substrate, wherein the thin film substrate comprises a first sideconstituting a floor of the first and second microchambers and a secondside opposing the first side, wherein the thin film substrate comprisesa thermoelectric sensor located under each of the first and secondthermally isolated microchambers and configured to measure thetemperature differential between the first and second thermally isolatedmicrochambers; wherein the thin film substrate includes a polymericdiaphragm made of a material having a glass transition temperaturegreater than 150° C. and thermal decomposition temperature greater than250° C.; wherein a flexural modulus of the thin film substrate is atleast about
 1. 2. The microdevice of claim 1, wherein the thermoelectricsensor includes at least one thermocouple, the thermoelectricsensitivity per thermocouple being greater than 80 μV/° C.
 3. Themicrodevice of claim 1, wherein the thermoelectric sensor is configuredas a thin layer thermopile including a plurality of elongated segmentsof dissimilar materials, adjacent segments of dissimilar materials beingjoined together at opposite ends, thereby forming thermocouplejunctions.
 4. The microdevice of claim 3, wherein the dissimilarmaterials include antimony and bismuth.
 5. The microdevice of claim 3,wherein the thin film substrate further comprises: a first microheaterand a first temperature sensor, each aligned under the first thermallyisolated microchamber; and a second microheater and a second temperaturesensor, each aligned under the second thermally isolated microchamber.6. The microdevice of claim 5, wherein the thermocouple junctions of thethermoelectric sensor are located near the center of each of the firstand second thermally isolated microchambers, and vertically aligned withthe first temperature sensor and the second temperature sensor,respectively.
 7. The microdevice of claim 5, wherein the thermopile islocated vertically away and insulated from each of the first and secondmicroheaters and the first and second temperature sensors.
 8. Themicrodevice of claim 1, wherein the material for the polymeric diaphragmis selected from polyimide, parylene, polyester, andpolytetrafluoroethylene.
 9. The microdevice of claim 1, furtherincluding a first introduction channel and a second introductionchannel, each configured to provide passive chaotic mixing for asolution flowing through the first or the second introduction channel.10. The microdevice of claim 9, wherein each of the first introductionchannel and a second introduction channel includes a portion having aserpentine shape.
 11. The microdevice of claim 9, wherein each of thefirst introduction channel and a second introduction channel includesinternal ridges sufficient for creating turbulence in the solutionflowing through the first or the second introduction channel.
 12. Themicrodevice of claim 9, wherein the solution includes a ligand and asample for isothermal titration calorimetry measurement.
 13. Themicrodevice of claim 1, wherein the flexural modulus of the thin filmsubstrate is at least about
 2. 14. A method of determining a thermalproperty of an analyte, comprising providing a microdevice, comprising:a first thermally isolated microchamber; a second thermally isolatedmicrochamber; a thin film substrate formed on a polymeric layer; whereinthe first thermally isolated microchamber and the second thermallyisolated microchamber are identical in volume and configuration, andarranged side by side, each supported on the thin film substrate,wherein the thin film substrate has a first side constituting a floor ofthe first and second microchambers and a second side opposing the firstside, wherein the thin film substrate comprises: a thermoelectric sensorlocated under each of the first and second thermally isolatedmicrochambers and configured to measure the temperature differentialbetween the first and second thermally isolated microchambers; a firstmicroheater and a first temperature sensor, each aligned under the firstthermally isolated microchamber; and a second microheater and a secondtemperature sensor, each aligned under the second thermally isolatedmicrochamber; and wherein the thin film substrate includes a polymericdiaphragm made of a material having a glass transition temperaturegreater than 150° C. and thermal decomposition temperature greater than250° C.; providing a thermal enclosure enclosing the microdevice;loading a sample material containing an analyte into the firstmicrochamber; loading a reference material into the second microchamber,the reference material does not contain the analyte; heating the thermalenclosure at a predetermined temperature scanning rate; determining athermal property of the analyte based on the measured temperaturedifferential between the first microchamber and the second microchamber;wherein a flexural modulus of the thin film substrate is at leastabout
 1. 15. The method of claim 14, further comprising providing atemporally periodic variation in the heating power during the heating ofthe thermal enclosure.
 16. The method of claim 14, wherein providing thetemporally periodic variation in the heating power comprises providingidentically temporally modulated heating to the sample material and thereference material by the first microheater and the second microheaterof the microdevice.
 17. The method of claim 16, further comprisingcalibrating the output of the thermoelectric sensor under a plurality ofmodulation frequencies.
 18. The method of claim 14, further comprisingcalibrating the output of the thermoelectric sensor using the first orthe second microheater of the microdevice that is aligned underneath thesample chamber to provide a constant differential heating power betweenthe first microchamber and the second microchamber.
 19. A method ofdetermining a thermal property of an analyte, comprising providing amicrodevice including two identically configured thermally isolatedmicrochambers, each of the two microchambers supported on a thin filmsubstrate formed on a polymeric layer, wherein the thin film substratecomprises a thermoelectric sensor configured to measure the temperaturedifferential between the first and second thermally isolatedmicrochambers; providing a thermal enclosure enclosing the microdevice;loading a sample material containing an analyte into one of the twomicrochambers; loading a reference material into the other of the twomicrochambers, the reference material does not contain the analyte;heating the thermal enclosure at a predetermined temperature scanningrate; providing additional temporally modulated heating to the samplematerial and the reference material during the heating of the thermalenclosure; and determining a thermal property of the analyte based onthe measured temperature differential between the first microchamber andthe second microchamber; wherein a flexural modulus of the thin filmsubstrate is at least about
 1. 20. A method of determining heat involvedin a reaction between at least two substances, comprising: providing amicrodevice, comprising: a first thermally isolated microchamber; asecond thermally isolated microchamber; a thin film substrate formed ona polymeric layer; wherein the first thermally isolated microchamber andthe second thermally isolated microchamber are identical in volume andconfiguration, and arranged side by side, each supported on the thinfilm substrate, wherein the thin film substrate has a first sideconstituting a floor of the first and second microchambers and a secondside opposing the first side, wherein the thin film substrate comprisesa thermoelectric sensor located under each of the first and secondthermally isolated microchambers and configured to measure thetemperature differential between the first and second thermally isolatedmicrochambers; wherein the thin film substrate includes a polymericdiaphragm made of a material having a glass transition temperaturegreater than 150° C. and thermal decomposition temperature greater than250° C., and providing a thermal enclosure enclosing the microdevice;providing a sample solution into the first thermally isolatedmicrochamber, the sample solution containing a mixture of a firstsubstance and a second substance at a first concentration ratio;providing a reference solution into the second thermally isolatedmicrochamber, the reference solution does not contain at least one ofthe first and the second substances; maintaining the thermal enclosureat a first predetermined constant temperature; determining the heatinvolved in the reaction between the first substance and the secondsubstance at the first ratio and the first predetermined constanttemperature based on the measured temperature differential between thefirst microchamber and the second microchamber; wherein a flexuralmodulus of the thin film substrate is at least about
 1. 21. A method ofdetermining heat involved in a reaction between at least two substances,comprising: providing a microdevice including two identically configuredthermally isolated microchambers, each of the two microchamberssupported on a thin film substrate formed on a polymeric layer, whereinthe thin film substrate comprises a thermoelectric sensor configured tomeasure the temperature differential between the first and secondthermally isolated microchambers; providing a thermal enclosureenclosing the microdevice; feeding a sample solution into the firstthermally isolated microchamber, wherein the sample solution is preparedmixing a first substance with a second substance; feeding a referencesolution into the second thermally isolated microchamber, the referencesolution does not contain at least one of the first and the secondsubstances; determining the heat involved in the reaction between thefirst substance and the second substance based on the measuredtemperature differential between the first microchamber and the secondmicrochamber, wherein a flexural modulus of the thin film substrate isat least about 1.